Fig. 1. Rapid generation of human GABAergic neurons by overexpression of Ascl1 and forskolin.
a Culturing paradigm for the generation of induced GABAergic neurons (iGABAA-FSK). b iGABAA-FSK neuron immunostaining at DIV 49 for neuronal marker MAP2 colabeled with Glutamate decarboxylase (GAD) 67 or GABA. c iGABAA-FSK are co-cultured from DIV 0 on with iGLUNgn2 to promote functional maturation (named E/I networks), in a ratio of E/I 65:35. d VGAT and Gephyrin co-localization in E/I networks at DIV 49. e Immunostaining for GABA colabeled with either calbindin (CB), calretinin (CR), somatostatin (SST), parvalbumin (PV), MEF2C (asterix), or synaptotagmin-2 (SYT2, arrowheads) in E/I networks (quantification sample size n = 7–9 coverslips per condition). f Heatmap showing expression of glutamatergic/GABAergic transporters and subtypes genes, and expression of genes important in GABAergic neuron development in E/I 65:35 networks at DIV 49 (three biological replicates from one neuronal preparation). Data represent the log-transformed counts per million (logCPM). g Representative firing patterns of iGABAA-FSK neurons at DIV 28, 35 and 49. Analysis of iGABAA-FSK membrane properties including h resting membrane potential (Vrmp) and i membrane capacitance (Cm). Analysis of action potentials evoked by step depolarization of iGABAA-FSK membranes including j fractions of maximum number of action potentials, and k Rheobase. l Quantifications of correlated synaptic input (number of synaptic burst/minute). m Spontaneous glutamatergic (red inset) and GABAergic (blue inset) postsynaptic inputs (sPSCs) received by iGLUNgn2. n Quantification of synaptic input types (DIV 28 n = 39, DIV 35 n = 38, DIV 49 n = 41 cells from three batches). All data represent means ± SEM. *p < 0.05; ***p < 0.001 (One-way ANOVA with Tukey correction was used to compare between DIVs). Scale bar is 20 µM, scale bars of zoom-in pictures are 6 µM.